An estimated two million Americans suffer from infections caused by multi-drug resistant (MDR) bacteria resulting in a substantial impact on patients' lives and an extraordinary economic burden. Due to the arsenal of antibiotic resistance mechanisms that these bacteria present, traditional antibiotic therapies are often ineffective. New strategies that use a novel mechanism of action are needed to augment the arsenal of therapeutic options to address the growing problem of MDR bacteria. Agile Sciences' co-founders, Drs. Christian Melander and John Cavanagh of NC State University, have developed a new class of 2- aminoimidazole (2-AI) small molecules that act via a novel mode of action to inhibit the ability of the bacteria to respond to environmental stimuli, thus rendering the bacteri more sensitive to antibiotics. The 2-AI molecules inhibit response regulator (RR) proteins of two-component systems resulting in lower antibiotic MIC values against MDR strains of Gram-positive and Gram-negative bacteria. In addition, the 2-AI compounds have favorable toxicity and metabolic stability profiles, and so they represent promising scaffolds for evaluation as potential therapeutics to address problems associated with MDR bacteria. The overarching goal of this proposal is to identify lead 2-AI molecules against each of three target bacteria (methicillin-resistant Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa) that substantially increase the efficacy of antibiotics and have pharmaceutically relevant attributes. RR proteins are an untapped target; therefore, there are insufficient tools fo target binding studies. For this reason, the objective of Aim 1 is to generate target binding assays against three RR proteins that have been implicated in antibiotic resistance (S. aureus VraR, A. baumannii PmrA, and P. aeruginosa CzcR). These binding assays, involving electrospray ionization mass spectrometry, surface plasmon resonance, and reporter strains, will be used to direct medicinal chemistry efforts in Aim 2. In addition to target binding, 2-AI derivatives synthesized in Aim 2 will be evaluated for MIC-lowering, cytotoxicity, metabolic stability, and plasma protein binding properties. This project will be overseen by Dr. Angela Pollard, Agile Sciences' Director of Research, who has successfully managed development programs at Agile Sciences. Dr. Cavanagh, an expert in bacterial cell signaling processes, will design and validate RR protein binding assays in Aim 1. Dr. David Jung, a medicinal chemist with 20 years of experience, and Dr. Steve Young, former head of Medicinal Chemistry at Merck, will be responsible for designing 2-AI derivatives in Aim 2. Dr. Jeff Collins, who has over 30 years of drug development experience specializing in anti-infectives, will provide consulting expertise. This project has the potential to significantly impact the field of antibiotic drug development. This new strategy for disabling bacterial resistance mechanisms could lead to a novel therapeutic that will provide clinicians with an effective treatment option for infection caused by MDR bacterial pathogens.